(2003) voltage-gated proton channels and other proton transfer pathways..pdf
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Voltage-Gated Proton Channelsand Other Proton Transfer Pathways
THOMAS E. DECOURSEY
Department of Molecular Biophysics and Physiology, Rush Presbyterian St. Lukes Medical Center,
Chicago, Illinois
I. Introduction 476II. Chemistry of Protons 477
A. Protons in solution: hydrogen bonds 477B. Proton conductance in water by the Grotthuss mechanism 478C. Proton transfer reactions 480
D. Proton transfer in the plane of the membrane: the antenna effect 480E. Control of pH 481F. Selected properties of buffers 483
III. Mechanisms of Proton Permeation Through Membranes 484A. Proton permeation through membranes without transport proteins 484B. Being and nothingness: do proton channels exist? 487C. Are proton channels real ion channels? 487D. Hydrogen-bonded chain conduction 490E. Proton transfer in water wires 492
IV. Classes of Proton-Permeable Ion Channels 493A. Gramicidin 493B. Normal ion channels 496C. Synthetic proton channels 497D. Aquaporins (water channels) 497E. M2viral proton channel 497
F. Fo, CFo, or Voproton channels of H
-ATPases 499G. Flagellar motor, MotA, MotB 501H. Bacteriorhodopsin 501I. Bacterial reaction center 502
J. Cytochromec oxidase 503K. Carbonic anhydrase 506L. Uncoupling protein of brown fat 507
M. Proton conductance associated with expression of various proteins with other jobs 507N. Summary of insights gained from other proton pathways 508O. Dependence of H current on H concentration (pH) 511
V. Voltage-Gated Proton Channels: General Properties 513A. What are voltage-gated proton channels? 513B. History 514C. Where are proton channels found? 515D. Varieties of voltage-gated proton channels 516
E. High proton selectivity 517F. Anomalously weak dependence ofgH
on H concentration 518G. Small unitary conductance 518H. Strong temperature dependence 519I. Large deuterium isotope effects 520
J. What is the rate-determining step in conduction? 521K. Voltage-dependent gating 522L. pH dependence of gating 524
M. Model of the mechanism of pH- and voltage-dependent gating 526N. Impervious to blockers 528O. Inhibition by polyvalent metal cations 530
VI. Voltage-Gated Proton Channels: Functions and Properties in Specific Cells 531A. Proton currents increase pH
irapidly and efficiently 532
Physiol Rev
83: 475579, 2003; 10.1152/physrev.00028.2002.
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B. Modulation by physiological mediators 533C. Excitable cells: snail neurons and skeletal myotubes 535D. Amphibian oocytes:Ambystoma and Rana esculenta 535E. Alveolar and airway epithelium 535F. Pulmonary smooth muscle: hypoxic pulmonary vasoconstriction 537G. Lymphocytes 537H. Phagocytes: macrophages, eosinophils, neutrophils, microglia 537
I. Molecular identity of voltage-gated proton channels: is part of the NADPH oxidase complex avoltage-gated proton channel? 548
J. Functional link between NADPH oxidase activity and H channel gating 553K. How far apart are proton channels and NADPH oxidase complexes? 553
VII. Summary and Conclusions 554
DeCoursey, Thomas E. Voltage-Gated Proton Channels and Other Proton Transfer Pathways. Physiol Rev 83:
475579, 2003; 10.1152/physrev.00028.2002.Proton channels exist in a wide variety of membrane proteins wherethey transport protons rapidly and efficiently. Usually the proton pathway is formed mainly by water moleculespresent in the protein, but its function is regulated by titratable groups on critical amino acid residues in the
pathway. All proton channels conduct protons by a hydrogen-bonded chain mechanism in which the proton hops
from one water or titratable group to the next. Voltage-gated proton channels represent a speci fic subset of protonchannels that have voltage- and time-dependent gating like other ion channels. However, they differ from most ion
channels in their extraordinarily high selectivity, tiny conductance, strong temperature and deuterium isotopeeffects on conductance and gating kinetics, and insensitivity to block by steric occlusion. Gating of H channels is
regulated tightly by pH and voltage, ensuring that they open only when the electrochemical gradient is outward. Thus
they function to extrude acid from cells. H channels are expressed in many cells. During the respiratory burst in
phagocytes, H current compensates for electron extrusion by NADPH oxidase. Most evidence indicates that the H
channel is not part of the NADPH oxidase complex, but rather is a distinct and as yet unidentified molecule.
I. INTRODUCTION
Voltage-gated proton channels are unique ion chan-
nels in several respects. They are called proton channels
because they behave like ion channels and are highly
selective for protons. Although protons exist in solutionalmost entirely in the form of hydronium ions, H3O, all
proton-selective channels conduct protons as H, rather
than H3O. This is true even for water-filled pores like
gramicidin. It remains a matter of some contention
whether proton channels should be considered to be ion
channels at all, although this designation seems more
appropriate than any alternative and is becoming ac-
cepted (444). Proton channels differ from carriers and
unequivocally are not pumps. Protons are unique ions
with respect to their behavior in bulk solutions, their
interactions with proteins, and the mechanism by which
they traverse ion channels and other molecules. The
unique chemical properties of protons explain why pro-ton channels hold the records for both the largest and
smallest single-channel currents. Thus there is an intro-
ductory discussion of selected aspects of proton chemis-
try. For a detailed discussion of the methods of pH mea-
surement, the reader is referred to the superb review by
Roos and Boron (850).
This review includes what I as a student of voltage-
gated proton channels consider to be useful and relevant.
Although the main focus is voltage-gated proton channels,
there is substantial coverage of salient properties of a
number of other proton-conducting molecules, for several
reasons. First, the structure and even the molecular iden-
tity of voltage-gated proton channels is essentially un-
known, whereas the structures of a number of other
proton-conducting molecules are known to within a few
Angstroms. Second, certain features that differentiateproton channels from other ion channels may be shared
among molecules whose function involves proton trans-
location. Once nature discovers a solution to a design
problem, this solution tends to recur (245). Proton con-
duction through the prototypical ion channel, gramicidin,
provides a frame of reference with respect to which we
interpret many results (deuterium and temperature ef-
fects, pH dependence, unitary conductance, etc.). It is
possible to distinguish two broad classes of proton-per-
meable molecules. Some molecules couple the flux ofprotons to a bioenergetic or enzymatic goal, such as
photosynthesis or CO2 hydrolysis. Other molecules aresimple proton channels that apparently exist for the sole
purpose of mediating protonflux across membranes. Inboth cases, proton flux is tightly regulated, either bycoupling to events central to the function of the molecule
or by a gating mechanism that turns protonflux on and offat appropriate times. A premise of this review is that the
molecular details of proton movement through all types of
proton-conducting molecules are likely to display similar-
ities with general applicability.
The properties common to all voltage-gated proton
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channels are described in detail. Then the properties and
proposed functions of H channels in specific cells arediscussed. There is a strong emphasis on proton channel
function in phagocytes, because much more is known
about function in these cells than in any other. Evidence
for and against the proposal that part of the phagocyte
NADPH oxidase complex functions as a proton channel(427) is summarized.
I do not expect more than a handful of people to read
the entire review. For those who study any of the numer-
ous molecules with proton pathways, I hope to present a
synopsis of their molecule from the vantage point of an
electrophysiologist interested in proton conduction. I feel
that it is useful to have information specifically regardingproton conduction in various channels/molecules assem-
bled in one place. Those who studynormalion channelsand are curious about H channels will want to know
their biophysical properties, which will appear esoteric
and tedious to others. Phagocyte biologists will be inter-
ested mainly in the section on H channels in phagocytes.For everyone else, the review should be a resource en-
abling a particular bit of information to be located in the
table of contents.
II. CHEMISTRY OF PROTONS
A. Protons in Solution: Hydrogen Bonds
Protons in aqueous solution almost always exist in
hydrated form as hydronium ions, H3O (or H3O
nH2O,
including waters of hydration), also called oxonium (605)or hydroxonium ions (1070). Protons exist as H1% of
the time during transfer from one water to another (184).
The three protons in H3O are equivalent, and each is
equally likely to jump to a neighboring water molecule
(84). The proton is unique among cations in being inter-
changeable with the protons that form water molecules.
This capability is significant in light of the tiny concentra-tion offree protons (H3O
) in physiological solutions,
40 nM, and the enormous total concentration of H in
water, 110 M. Only one proton in a billion is part of H3O
at any moment. The average lifetime of the H3O ion is
1 ps in liquid water at room temperature: estimates in
chronological order include 0.65 ps (84), 0.24 ps (184), 3.0ps (287), 1.7 ps (636), 1.1 ps (11), 1.3 ps (1095), 0.95 ps
(1050), and 0.5 0.79 ps (890). The proton is also unique asa monovalent cation in having no electrons, giving it a
radius 105 smaller than other ions, which greatly facili-
tates proton transfer reactions (80) and electrostatic in-
teractions with nearby molecules (696).
The quintessential feature of water and other proton
conduction pathways is the hydrogen bond (80, 84, 287,
361, 380, 469, 470, 592, 605, 799, 800, 967, 1101). Huggins
appears to have originated the concept of the hydrogen
bond while in the laboratory of Latimer and Rodebush.
Huggins conceived the idea of a hydrogen kernel heldbetween two atoms in organic compounds, which he did
not publish until 1922 (468); several earlier investigators
discussed interactions that in retrospect could be consid-
ered examples of hydrogen bonds (490). In 1920, Latimer
and Rodebush (592) adopted this idea and applied it towater, foreseeing the existence of networks of water
molecules, and used hydrogen bonding to explain the high
mobility of protons in water asa sort of Grotthuss chaineffect, rather than . . . a rapid motion of any one H3O
ion.
Water . . . shows tendencies both to add andgive up hydrogen, which are nearly balanced. Then, interms of the Lewis theory, a free pair of electrons onone water molecule might be able to exert sufficientforce on a hydrogen held by a pair of electrons onanother water molecule to bind the two moleculestogether. Structurally this may be represented as
Such combination need not be limited to the forma-tion of double or triple molecules. Indeed, the liquidmay be made up of large aggregates of molecules,continually breaking and reforming under the influ-ence of thermal agitation. Such an explanation
amounts to saying that the hydrogen nucleus heldbetween 2 octets constitutes a weakbond 1 (592).
Linus Pauling coined the term hydrogen bond in a
general paper on chemical bonds (798) and developed
and popularized the idea in a chapter of his book, The
Nature of the Chemical Bond (800).
Water molecules tend to form tetrahedral hydrogen
bonded structures, at least ideally (84). In ice the tetra-
hedral structure exists (799) and is evidently so rigid at
very low temperature (i.e., the dielectric constant drops
drastically) that proton conduction is limited (188, 261,
313). In liquid water, however, the tetrahedral ideal is not
achieved, and the actual coordination number decreaseswith increasing temperature (300, 366), which likely ac-
counts for the greater decrease in activation energy at
higher temperatures for proton transport than for other
ions (319, 605, 784, 786). Water can be considered a
broken down ice structurewith continual formation andbreaking of hydrogen bonds (707). Although protons in
1 M. Huggins of this laboratory, in some work as yet unpublished,has used the idea of a hydrogen kernel held between two atoms as atheory in regard to certain organic compounds (592).
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water are formally considered to exist as H3O mole-
cules, it has long been recognized that larger molecular
groupings exist and are central to the understanding of
proton conduction. As early as 1936, Huggins (470) ex-
plicitly postulated the existence of H5O2, showed that
proton conduction can occur by shifts in the identities of
the water molecules that comprise the cation, and sug-gested that the rapidity of such shifts accounts for the
high mobility of protons in water. The two main larger
species are the so-called Zundel cation, two waterssharing an excess proton as H5O2
(470, 1102, 1103), and
theEigen cation, four waters sharing an excess protonas H9O4
(80, 287, 1070), although a transitional H13O6
structure has also been proposed (1049). These quasi-
molecules are in a sensefictitious, in that they are ideal-izations that exist only transiently along with many unde-
fined intermediate or alternative states (664, 890). Quan-tum molecular dynamics simulations show that a proton
in water sometimes shuttles back and forth between two
neighboring water molecules many times per picosecond,behavior that defines a Zundel (or Huggins) cation, butalso spends time associated with a single water (which is
hydrogen bonded to threefirst shell waters) as an Eigencation (890, 1050). Eigen thought that the proton in H9O4
was essentially delocalized (288) and shared among three
of the waters surrounding the H3O molecule; the fourth
water is oriented incorrectly for rapid proton transfer
(605). Ab initio molecular dynamics calculations indicate
that a proton in water is affiliated with one oxygen atomas H3O
(H9O4, including the primary hydration shell)
60% of the time, and 40% of the time it is intermediate
between two oxygens as H5O2
(1025). Although the pro-ton spends blocks of time as H9O4 (i.e., associated with a
single oxygen), these events occur within bursts of oscil-
lations between the same pair of oxygens as though the
proton remembers its former partner (1050), and hence,
appearances to the contrary, was never truly delocalized.
B. Proton Conductance in Water by the
Grotthuss Mechanism
That there is a fundamental difference between pro-
tons and other cations is clear from the fivefold higherconductivity of H in water than other cations like K (84,217). In fact, considering its degree of hydration (based on
solution density) H might be expected to have a low
mobility like Li (84, 845) but has nine times higher
mobility (845). It has long been appreciated that protons
are conducted by a special mechanism in which they hop
from one water molecule to the next, which is often called
the Grotthuss mechanism, although de Grotthuss pro-posal (254a) differs from current views. The Grotthuss
mechanism is also called prototropic transfer (605), todistinguish it from ordinaryhydrodynamic diffusion of
H3O as an intact cation. Danneel (217) suggested that a
proton in an electric field might bind to one side of awater molecule and that another proton could leave the
far side of the molecule, thus saving the time it would
have taken to diffuse that distance. A key distinction from
other ions is that during proton conduction the identity of
the conducted proton changes (84). Except for Huckelstheory (467a), the equivalence of the three protons inH3O
is generally considered to be essential to the special
prototropic conduction mechanism. Danneel further pro-
posed in 1905 (217) that proton conduction by a Grotthuss
mechanism requires two processes: proton hopping from
one water molecule to the next, and also a reorientation
of water molecules. Glasstone, Laidler, and Eyring (366)
concluded that proton transfer was rate-limiting and that
water rotation was rapid. Conway, Bockris, and Linton
(184) concluded that the proton transfer step was rapid
and proposed that the rate-determining step was the re-
orientation of the recipient water molecule in the electri-
calfield of the donor H3O (184, 448). More recent theo-ries growing out of Eigen and co-workers views agreethat the proton transfer step is rapid, but ascribe the
rate-limiting step to reorganization of the hydrogen-
bonded network through which H conduction occurs
(10a, 11, 221, 479, 664, 1024, 1025, 1050).
The special prototropic conduction mechanism ap-
pears to require a hydrogen-bonded structure (361, 469,
605). Water is an ideal medium for prototropic conduction
because of its propensity to form hydrogen bonds; water
has a higher viscosity compared with other solvents due
to hydrogen bonding (300). Proton conduction occurs
essentially by means of changes in the identity of thewater molecules that participate in the hydrogen-bonded
network that includes the excess proton. The mechanism
of proton conduction in water has been described as
structural diffusion, which was felt to reflect the delo-calized nature of the solvated proton within a hydrogen-
bonded network (287, 319, 1070). The concept of struc-
tural diffusion of protons in water is supported by ab
initio molecular dynamics simulation (1024). Proton con-
duction occurs as a result of isomerization between Zun-
del and Eigen cations (10a, 11, 1024). The rate-determin-
ing step appears to be the breaking of a second shell
hydrogen bond, which allows the replacement of one of
the waters by a different one (10a, 287, 288, 664). Thisprocess has been called the Moses mechanism, withsecond shell hydrogen bonds breaking in the path of the
proton and reforming behind (10a, 11a), just as the Red
Sea parted to allow Moses and his companions to cross
(Exodus 14:2127). At this point the modern view (10a)diverges from most earlier models in which the water
molecule immediately adjacent to H3O is required to
rotate into an appropriate configuration to accept theproton (80, 184, 448, 467a). The threefirst shell hydrogenbonds are too strong to be easily broken (10a), whereas
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the second shell hydrogen bonds are expected to be of
normal strength, 2.6 kcal/mol, consistent with empirical
measurements of proton mobility (636, 678). In Agmonsview, the widely used traditional method of estimating the
abnormal component of H mobility by subtracting themobility of anormalcation such as Na or K from the
total H
mobility (319, 361, 366, 467a, 605, 628, 636, 678,845) is incorrect. Because the H3O
ion is tightly hydro-
gen bonded to its first shell neighbors, it is effectivelyimmobilized. Consequently, essentially all of the mobility
of protons in solution is of the abnormal (Grotthuss type)
variety (11). Another difference is that in contrast to
Eigens delocalized proton that could move freely withinthe H9O4
complex (287, 319, 1070), in the current view
the proton is mainly associated with a single oxygen or
vascillates rapidly between two oxygens, and eventually
transfers successfully as a result of second shell hydrogen
bond rearrangement (10a, 890, 1024, 1025, 1095).
Because waters inside proton channels may be
bound or constrained in some way, proton movementthrough water-filled channels is often considered to bemore analogous to proton transport in ice than in water
(732, 733). Proton conduction in ice is fundamentally
different from that in liquid water (288, 552, 771, 783). The
extensive hydrogen bond rearrangement that character-
izes proton transfer in water cannot occur in ice (552,
771). Liquid water is mainly three-coordinated, but the ice
structure enforces four-coordination. Repulsion from the
fourth water pushes the H3O closer to its neighbors,
decreasing the energy barrier for proton transfer (552,
771). Historically, the question of proton conduction in ice
has proven to be difficult and controversial (42, 44, 96,157, 188, 288, 294, 380, 500, 808). Eigen and colleaguesreported that the mobility of H in ice was extremely high
(289), 12 orders of magnitude higher than in water (288),and differingfrom that of conduction band electrons inmetals by only about 2 orders of magnitude (287). Sub-sequently, the general consensus has been that these
measurements were contaminated by conduction through
melted water at the surface and that the true mobility is
much lower, 3 104 to 6.4 103 cm2 V1 s1,
typically 103 cm2 V1 s1 (142, 157, 294, 575, 734,
782, 808, 809). The mobility of H in water is 3.6 103
cm2 V1 s1 (845). It is a major problem to determine
the number of defects (ionic or bonding) in ice, whichmust be known to calculate mobility. Pure ice almostinvariably contains enough impurities to dominate at-
tempts to measure the mobility of ionic defects, which are
present at only 1 per 1013 H2O molecules at 20C(808). This problem can be overcome bydoping the icewith carriers so that their concentration is known and the
signal is larger and thus more accurately measurable
(380). In ice studies, it is important to distinguish events
at the surface from events occurring within the bulk
phase, although the former can be useful in dissecting
elementary processes that contribute to proton mobility
(260, 356, 357, 1028, 1083).
The only ions that carry current in ice are H and
OH, and both move as a consequence of proton or
proton defect movement (783). Both protons and Bjerrum
defects (see sect. IIID) must move for sustained current
(380); movement of L defects (or protons) alone simplyproduces (or eliminates) polarization (782). In pure ice at
moderate temperatures, the dominant charge carrier is
the Bjerrum L defect (the conduction of which occurs by
rotation of water molecules), and thus for DC conduction
the motion of the ionic defect (H3O) is rate determining
(809). Protons tend to become shallowlytrappedby themore abundant Bjerrum L defects, but above 110 K they
escape at a significant rate and are mobile until theyencounter the next trap (1083). Data on H3O
soft-landed onto the surface of ice were interpreted to meanthat at temperatures below 190 K proton conductance in
ice is essentially absent (188). One danger that must be
considered in such studies is that protons can be trappedat the ice surface (1028), probably because the 4-coordi-
nated state that is enforced inside ice is less favorable
than the less stringent coordination at the surface (552).
Earlier studies of isotope exchange in pure and in doped
ice had indicated that Bjerrum defect and proton migra-
tion occurred to a similar extent in ice in the 135 150 Krange, although OH lacked mobility (181). A recent
study of isotope exchange in pure ice nanocrystals at
145 K revealed clear evidence of mobility of both Bjerrum
L defects and protons, based on the distinctive infrared
spectra of D2O, coupled HDO molecules, and isolated
HDO (1028). Most evidence indicates that protons aremobile in ice at least down to 110 K (1083), and possibly
as low as 72 K (808), that proton mobility in ice is prac-
tically temperature independent (782, 808), and that the
mobility of H3O at 100 K is within an order of magni-
tude of that in liquid water (808).
The hydroxide anion (OH) also has anomalously
high conductivity compared with other anions, 198 cm2
S/eq (218, 845, 943), although not quite so extreme as H
at 350 cm2 S/eq (786, 845, 921). In addition, the activa-
tion energy for OH conductivity is higher than for H
(288, 623, 636). The high mobility is believed to reflectOH migration by a Grotthuss-like mechanism in which
the OH moves from one water to the next by virtue of aproton hopping in the opposite direction (80, 84, 184, 217,
469, 605, 845). Protons move via prototropic transfers
between H3O and H2O, whereas OH
migrates by pro-
totropic transfers between H2O and OH. The rate-deter-
mining step in OH mobility may be the same as for H
mobility, the breaking of a second shell hydrogen bond
(11b), although a recent proposal invokes the crucial
breaking of afirst-shell hydrogen bond (1026). That OH
mobility is less than H mobility in spite of the similarity
of mechanism has been explained in several ways. Bernal
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and Fowler (84) proposed that the two protons in the
donor H2O are held more tightly than the three protons in
the donor H3O molecule, thus reducing the likelihood of
the former proton transfer. Conway et al. (184) felt the
critical difference was the electrostatic facilitation by the
extra proton in H3O of the prerequisite and rate-limiting
water rotation that precedes proton transfer. Gierer andWirtz (361) suggested a charge mechanism: for H trans-
fer the proton hops between neutral H2O molecules,
whereas for OH the proton hops between two residual
negative charges (288, 361). Agmon (11b) proposed that
contraction of the O-O bond distance adds an extra 0.5
kcal/mol to OH transfer. Onsager proposed that H mo-
bility is higher because the additional kinetic energy of
the excess proton increases the energy of H3O and fa-
vors subsequent proton transfer, whereas in OH conduc-
tion the energy of the proton is transferred from OH to
H2O and thus does not contribute to the next transfer
(J. F. Nagle, personal communication).
C. Proton Transfer Reactions
Eigen (287) studied proton transfer reactions exten-
sively and formulated general rules that govern such re-
actions. Proton transfer reactions tend to be very rapid
and are described as diffusion controlled because therate of the reaction is determined by the frequency of
molecular encounters resulting from diffusion (287). The
rate of proton transfer in normal proton transfer reactions
depends on the pKadifference between donor and accep-
tor, as illustrated in Figure 1.2 When pKacceptor pKdonor,
the forward reaction is rapid and independent of the pKadifference. Protonation of various bases occurs with a
rate constant 1010 M1 s1, with the electrostatically
favorable recombination of H and OH clocking in at
1.4 1011 M1 s1 (287). When the forward reaction is
diffusion controlled, the reverse reaction will occur at a
rate that is linearly related to the pKdifference (Fig. 1A).
By definition, log kf log kr pKacceptor pKdonorpK(290). If the reaction is asymmetrical with respect to
charge (e.g., HX Y X HY), then the diffusion-
controlled limit will be different for the forward and
backward reactions (Fig. 1B). A Bronsted plot (123a)
provides similar information (787). A more thorough the-oretical development of the kinetics of proton transfer
invokes Marcus rate theory (654), as has been applied
successfully to carbonic anhydrase (931).
In terms of a proton conduction pathway that is
composed of a series of protonation sites, proton hops
may not obey the same rules as proton transfer reactions
in diffusion-controlled reactions, due to steric con-straints, etc. However, the general principles of the pK
dependence of transfer rates are likely to apply. Contin-
uous prototropic transfer is most efficient when the donorand acceptor are symmetrical, as in water to water trans-
fer (605). In solvent mixtures, the solvent with higher
affinity traps the proton (605). Ab initio molecular orbitalmethod calculations indicate that in a long water wire,
multiple proton transfers (hops) can occur simulta-
neously (i.e., energetically coupled to each other) using
the energy cost associated with a single transfer event
(882). An example of coherent proton tunneling has been
observed directly in a network of four coupled hydrogenbonds (465).
D. Proton Transfer in the Plane of the Membrane:
The Antenna Effect
There is long-standing debate over the suggestion
that protons may diffuse laterally at the surface of the
membrane at a higher rate than they diffuse in bulk
solution. The question has been discussed extensively in
2 The concept of pKa was introduced by Hasselbalch (415) actingon a suggestion by N. Bjerrum.
FIG. 1. Idealized dependence of the normalized rates of protontransfer reactions on the pKa difference between donor and acceptormolecules participating in the reaction. InA, the transfer is symmetricalwith respect to charge (e.g., HX Y X HY), whereas in B , thereaction results in charge neutralization. The slopes of the forwardreaction () and backward reaction () limit at 0 or 1 at large pK. Thelimiting rate constant (kmax) is 10
9 to 1010 M1 s1 for a diffusion-controlled reaction. [From Eigen and Hammes (290), copyright 1963
John Wiley & Sons, Inc. Reprinted by permission of John Wiley & Sons.]
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the context of bioenergetic membranes (404, 418, 527,
530, 706, 724, 731, 820, 821, 1002, 1079). This question has
arisen in several instances in which the apparent single
proton channel current is larger than the maximum rate at
which protons can diffuse to the channel, as predicted by
simple diffusion models. To some extent, surface en-
hancement may be ascribed to geometric factors, i.e., thedifference between diffusion in two and three dimensions
(353) without specifying the mechanism by which protons
would bind to the surface. A proton trapped at the mem-
brane surface will diffuse into a proton channel if it does
not first desorb, whereas a proton in three-dimensionalbulk solution has a low probability of diffusing into the
channel. In unbuffered solutions, surface conduction
dominates; in buffered solutions, the dominant pathway
depends on protonated buffer concentration and the ef-
fective size of the proton collecting antenna (353) (see
below).
One general way that surface conduction could en-
hance protonfluxes through a channel is by the antennaeffect (400, 867). Rather than requiring a proton to dif-fuse directly to the channel entrance, the entire mem-
brane surface, by virtue of its many negatively charged
groups, might collect protons, which then travel in the
plane of the membrane surface to the channel. Detailed
experimental and computational studies have been done
on this question (155, 353, 400, 653, 867). Protonation
reactions are often extremely rapid and limited only by
diffusion, with rate constants typically 1 6 1010 M1 s1 (287, 290, 400, 653, 867). One of the most rapid reac-
tions known is the recombination of H and OH with a
rate constant 1.4 10
11
M
1s
1
(287). However, occa-sionally higher rate constants are observed. An anoma-
lously high protonation rate measured for a site on a Ca2
channel, 4 1011 M1 s1, was explained by proposing
the site to be negatively charged and located in the chan-
nel vestibule, which would funnel the electric field linesand enhance the electrostatic attraction (823). If two
negatively charged groups (e.g., at the surface of a mem-
brane) are close enough together that their Coulomb
cages overlap, the virtual second-order rate constantgoverning the transfer of a proton from one group to the
other can be 1012 M1 s1 or greater (400), with the
current record being 6 1012 M1 s1 (867). The prob-
ability that a proton that is bound to a site with 1 chargeat the interface between membrane and aqueous solution
will transfer to a neighboring site, also with 1 charge,
rather than entering bulk phase, calculated with the De-
bye-Smoluchowski equation, is close to 100% for a 12-separation, decreasing with distance to 40% for a 60-separation (867). It seems clear that rapid proton transfer
in the plane of the membrane is possible.
On the other hand, the extent to which rapid surface
conduction might play a significant role must be estab-
lished in each specific situation. In a study on protontransfer rates between superficial amino acid groups ontuna cytochromec oxidase, all of the virtual second-order
rate constants were 109 except for one that was as large
as 1011, which was between groups within 10 of eachother (652). A cluster of three carboxylates on bacterio-
rhodopsin acts as a proton-collecting antenna, each witha high protonation rate of 5.8 1010 M1 s1, but the
dimensions of the antenna are smaller than those of the
molecule. Long-range proton migration occurs along a
protein monolayer, but depends critically on molecular
packing, and is abolished at low or high protein densities
(331). Molecular dynamics simulation indicates that pro-
ton transport near the surface of a dipalmitoylphosphati-
dylcholine membrane is inhibited rather than enhanced
(953). Finally, de Godoy and Cukierman (253a) explored
the effects of bilayer composition on H currents through
gramicidin channels. The limiting H conductance at low
pH was the same in bilayers formed from protonatable
phospholipids that presumably should be capable of me-diating lateral H conduction and bilayers formed from
covalently modified phospholipids that cannot be proton-ated. Furthermore, differences in the H conductance at
higher pH were fully accounted for by electrostatically
induced changes in local H concentration near the mem-
brane, providing no evidence of significant lateral H
conduction (253a). In summary, it appears that rapid pro-
ton transfer at the membrane surface may occur under
specialized conditions but cannot be assumed to occur
generally.
E. Control of pH
The usual way to control pH is with buffered solu-
tions. Because the control of pH is never perfect, recog-
nizing systematic sources of error is useful. Voltage-gated
proton channels appear to be perfectly selective for pro-
tons over all other ions besides deuterium, as discussed in
section VE, and hence act as local pH meters (237). Se-
lectivity is evaluated by measuring the reversal potential
(Vrev) in solutions of various pH, and comparing the result
with the Nernst potential for H (EH)
EHRTF log H
oHi
(1)
Although reasonable agreement between the measured
Vrev and EH is often obtainable, the agreement is rarely
perfect. If we tentatively accept the conclusion that volt-
age-gated proton channels are perfectly H selective (see
sect.VE), then any deviation ofVrevfromEHindicates that
the true pH differs from the nominal pH. The primary
cause of this deviation in patch-clamp experiments is
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imbalance between the rate that proton equivalents cross
the cell membrane and the rate the buffer from the pipette
replenishes the cytoplasmic compartment. The intracellu-
lar compartment is a large unstirred volume, and proton
efflux such as that occurring during H currents willdeplete protonated buffer from the cell. For example, a
10-m-diameter cell has a volume of 524 fl, and if it isfilled with a pipette solution that has 100 mM buffer at itspKa, the entire cell will contain 1.6 10
10 protonated
buffer molecules. During a modest sustained outward H
current of 100-pA amplitude, 6.25 108 H leave the cell
each second, deprotonating 4% of the total protonated
buffer. Even at intracellular pH (pHi) 6 there are only
315,000 free protons in the entire cell, all of which would
be consumed during 0.5 ms of H current. Thus, essen-
tially the entire H current is carried by protons that
immediately previously were bound to buffer molecules.
Replenishment of buffer occurs by diffusion from the
pipette solution and requires the diffusion of these rather
large molecules through a small 1-m-diameter pipettetip into the cell.
Calculations based on Pusch and Nehers empiricaldetermination of diffusion rates (827) predict a time con-
stant of 19 s for the equilibration of 250-Da buffer mole-
cules from a pipette with 5-M tip resistance into a
15-m-diameter cell. This time constant is proportional to
cell volume (776). The rate of equilibration of pHi will be
slower than that for simple buffer diffusion, due to the
effective slowing of H diffusion by fixed (immobile)intracellular buffers (514). Direct estimates of the time
constant of equilibration of pHi in HL-60 cells and macro-
phages of unspecified size were 11 s (258) and 58 s or 97 s(519), respectively, representing at least qualitative agree-ment.
The presence and action of any membrane trans-
porter that moves proton equivalents across the cell mem-
brane will alterVrev. Thus, when Na is present only in the
external solution and pHiis low, the inward Na gradient
and outward H gradient both conspire to activate
Na/H antiport. H extrusion by the antiporter is rapid
enough to raise pHisubstantially (i.e., by 0.5 unit or more)
in alveolar epithelial cells studied in whole cell patch-
clamp configuration, in spite of the presence of 119 mMbuffer in the pipette solution (237). H is extruded by the
antiporter faster than the supply is replenished by diffu-sion of protonated buffer from the pipette. Geometrical
factors influence this balance, with smaller cells or largerpipette openings attenuating the change in pHi due to
antiport activity. Thus manifestations of Na/H antiport
were less pronounced in human neutrophils (237) or mu-
rine microglia (546) than in the larger rat alveolar epithe-
lial cells, but obviously differences in the expression of
Na/H antiport molecules could also play a role. Any
other mechanism that results in net movement of H
equivalents across the membrane will alter pHi. Several
mechanisms of membrane H flux are discussed in sec-tion IIIA, of which the shuttle mechanism in particular
could cause attenuation of the pH gradient across the
membrane (see sect. IIIA3).
A systematic deviation arises whenVrev is measured
by the conventional tail current protocol. A depolarizingprepulse activates the H conductance (gH) and then the
voltage is repolarized to various levels, and the direction
of the tail current (the decaying current waveform that
reflects the progressive closing of H channels) is ob-served. The necessity to activate a substantial gH during
the prepulse to elicit an interpretable tail current, com-
bined with the extremely slow activation kinetics of volt-
age-gated proton channels in mammalian cells, inevitably
causes significant depletion of intracellular protonatedbuffer during the prepulse. If a comparable H current is
elicited during the prepulse in solutions of varying pH, the
error will be a relatively constant addition of a few milli-
volts to the measured Vrev. This systematic error mayexplain why the vast majority ofVrevmeasurements in the
literature are more positive than EH. On the other hand,
Vrevmeasurements that encompass negative pH [pHi
extracellular pH (pHo)] indicate deviation in the opposite
direction in this range (166, 519, 886), suggesting that an
element of dissipation of any pH gradient may also play a
role. As a result, measurement of the change in Vrev at
several pH rather than the absolute Vrevoften provides a
cleaner estimate, which explains the fondness that many
experimentalists have for this way of expressing their
data. Direct measurements of Vrev using prepulses that
elicit smaller or larger currents have been shown to raisepHi and hence shift Vrev positively roughly in proportion
to the integral of the outward H current during the
prepulse (70, 232, 372, 473, 519, 709), although this effect
is not apparent in large cells (134). It is important to
recognize that the deviation of Vrev from EH is not an
error, but instead accurately reflects the effects of thepulse protocol on pHi. We consider voltage-gated proton
channels to be perfect pH meters (see sect. VE).
An expedient way to estimateVrev is to activate the
gH and then ramp the membrane voltage downwardfrom positive to negative (372). If enough channels open
at positive voltages and the ramp is rapid enough that the
channels remain open, then Vrevcan be taken as the zerocurrent voltage, although any leak conductance and ca-
pacity current must be either negligibly small or cor-
rected. The problem remains that it is first necessary toactivate the gHto observe Vrev, so this approach does not
avoid the problem of depletion. Another clever way to
estimate Vrev is simply to interpolate between the H
current at the end of a depolarizing pulse and that at the
start of the subsequent tail current (473). One required
assumption is that the instantaneous current-voltage re-
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lationship be approximately linear. This method is useful
in certain situations, particularly if one suspects that sig-
nificant depletion has occurred. The advantage is thatboth required data points are obtained by applying a
single pulse, and they are measured at nearly the same
time. Again, this approach does not avoid the effects of
depletion. In fact, its originators used this approach todemonstrate that H efflux during large depolarizing
pulses alkalinized the cytoplasm significantly.H currents increase pHiin proportion to the amount
of H extruded. For small currents, the change in pHimay
be negligible, but for large currents, depletion of proton-
ated buffer will noticeably increase pHi. These effects are
less pronounced in large cells (134) because they reflectthe area-to-volume ratio. Restoration of pHiis determined
by the geometrical factors already discussed, and typi-
cally requires tens of seconds up to several minutes. A
useful rule of thumb is that because voltage-gated proton
channels do not inactivate, when the H
current peaksand then droops during a sustained depolarization, this
always reflects an increase in pHi. Experimentally, thisphenomenon can be annoying, but it is simply a manifes-
tation of the ability of the H conductance to do its job,
namely, to extrude acid at a rate adequate to alkalinize the
cytoplasm rapidly.
Perhaps not surprisingly, variations in extracellular
buffer from 1 to 100 mM had very little effect on voltage-
gated proton currents (241). The bath solution represents
an effectively infinite sink for protons. The situation forintracellular buffer is more complicated. Several whole
cell patch-clamp studies in which pHi was determined
have revealed that including 510 mM buffer in the pipettesolution does not control pHi as well as higher buffer
concentrations, e.g., 100 120 mM (232, 258, 519, 574). Inaddition, the time course of the H current during a single
depolarizing pulse was shown to depend strongly on in-ternalbuffer concentration in excised inside-out patchesof membrane (241). The initial turn on of H current was
similar, but the longer the pulse, the more the current
with 1 mM buffer drooped relative to that with 10 mM
buffer. Nevertheless, decreasing internal buffer from 100
to 1 mM attenuated the H current by only 50%; thus
this effect is attributable to H current-associated pH
changes, rather than a limitation of the conductance ofthe channel by buffer (241) (cf. sect. VJ).
In addition to buffers, application of an NH4 gradient
has proven to be a useful way to control pHi in patch-
clamped cells (242, 248, 387) (see also sect. IIID). Control
over pHi is excellent and rapid when the NH4 gradient is
symmetrical, becoming less effective for large NH4
(hence pH) gradients (248, 387). An advantage of this
technique is that pHi can be changed in a cell simply by
altering the bathing solution.
F. Selected Properties of Buffers
Several issues related to buffers are relevant to the
study of proton channels. Experimental control of pH
requires adequate buffering, as just discussed in section
IIE. Buffering power (or buffering capacity) is defined as
dB/dpH (1036), i.e., the concentration of strong base re-quired to change the pH of a solution by one unit. A more
rigorous discussion of this and other definitions can befound elsewhere (849, 850). The reported buffering power
of the cytoplasm in mammalian cells ranges from 18 to 77
mmol pH1 liter1 (850). The measured buffering
power of most cells increases substantially at lower pH,
typically three- tofivefold between pHi7.5 and pHi6.5 (24,41, 92, 324, 603, 630, 840, 850, 1067). A similar observation
has been made for the Golgi (153). The buffering power is
maximal at the pKaof the buffer (425, 1064), where it is
(ln10)[B]/4 0.58[B], where [B] is the total buffer con-
centration (559, 849, 1036). Thus a cytoplasmic buffering
power of 58 mmol pH1 liter1 would reflect thepresence of the equivalent of at least 100 mM simple
buffer in cytoplasm. To control pH experimentally, many
investigators use solutions with 100 mM exogenous buffer
near its pKa. Under normal conditions, this is adequate to
prevent pH changes large enough to alter H currents
noticeably (240) (but see cautionary tales in sect. IIE).
When a cell is dialyzed with a pipette solution con-
taining inadequate buffer, intrinsic cytoplasmic buffers
override the attempts of the pipette solution to control
pHi. The larger the cell, the more difficult is the control ofpHi. Byerly and Moody (135) compared the rate of equil-
ibration of pipette solutions containing K
or highly buff-ered H with cytoplasm in large neurons (90 120 m indiameter) studied with suction pipettes one-third the cell
diameter. The effective equilibration of H even with high
buffer concentrations (50100 mM) was three to fivetimes slower than that of K, and with 20 mM buffer, little
control over pHi was achieved (135). Similarly, the effec-
tive diffusion coefficient of H in cytoplasm isfive timesslower than that of mobile buffers (15). In small cells
studied with patch pipettes containing pH 5.5 solutions,
pHi deduced from the Vrev of H currents was 5.7 for
119 mM MES buffer and 6.3 for 5 mM MES (232). A
pipette solution with 1 mM buffer appeared to have es-
sentially no effect on pHi (240).Buffers have variable tendencies to chelate metal
ions (805). Because we could not find much informationon this property for normal pH buffers beyond the initial
description of the Good buffers (370), we measured the
binding constants of several buffers for Zn2, Cd2, Ni2,
and Ca2 (163). Certain buffers bind Zn2 avidly, includ-
ing tricine and N-(2-acetamido)-2-iminodiacetic acid
(ADA). The latter has been used to establish free Zn2
concentrations in the nanomolar range (22, 792).
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III. MECHANISMS OF PROTON PERMEATION
THROUGH MEMBRANES
A. Proton Permeation Through Membranes
Without Transport Proteins
In addition to the plethora of membrane proteinswhose function is to transport protons or acid equivalents
across cell membranes, there are several mechanisms by
which protons can permeate phospholipid membranes in
the absence of proteins. These mechanisms will be con-
sidered in part in the context of deciding whether voltage-
gated proton channels really exist or if they might simply
reflect one of the several nonprotein mechanisms of con-duction. A large literature exists on the proton permeabil-
ity of the cell membrane itself (see sect. IIIA1), largely
with respect to the important bioenergetic systems in
which large proton gradients are created. Thus, in mito-
chondria, chemical energy is stored as a proton gradientthat drives ATP generation. In chloroplasts, light energy is
transduced into a proton gradient to create ATP. Energy
transduction thus requires the generation of large proton
gradients. Nevertheless, many studies indicate that the
proton permeability of cell membranes is much higher
than that of other cations.
The Born self-energy cost of an ion permeating a pure
lipid bilayer is prohibitive (794), 58.6 kcal/mol for the
H3O (243). Therefore, a mechanism subtler than brute
force is required to translocate protons across mem-
branes. Four mechanisms that have been proposed to
explain proton permeation through biological membranes
are as follows: transient water wires (sect. IIIA2), weakbase or acid shuttles (sect. IIIA3), phospholipid flip-flop(sect. IIIA4), and specific proteins (channels, carriers, and
pumps) whose function is to transport protons. Highin-trinsic proton permeability must be explained by one ofthese mechanisms. As will become apparent however, the
proton permeability of cell membranes that contain volt-
age-gated proton channels is several orders of magnitude
higher than the highest estimate for simple phospholipid
bilayers. In most cells with H channels, any proton
permeability of the membrane itself is negligible in com-
parison (242).
1. Intrinsic proton permeability
It has been maintained widely and for some time that
membrane proton permeability (PH) is anomalous in two
respects. First, PH is many orders of magnitude higher
(104 to 102 cm/s) than the permeability of other cations
(1012 to 1010 cm/s) (227, 228, 390, 755, 797). Second, the
proton conductance (GH) is practically independent of pH
(226, 395, 396, 755). These observations have been chal-
lenged on various counts, and some of the complications
will be mentioned here.
PH is difficult to measure, and reported values varyover many orders of magnitude, ranging from 109 to
101 cm/s (153, 396, 585, 688, 755, 764, 766, 797, 804).
Although various studies report no (124), moderate (585),
or strong (i.e., up to 100-fold) (228, 390, 396, 755, 764,
804, 1033) dependence of PH on the composition of the
membrane, this dependence does not come close to re-solving the vast disparity in reported values. The idea that
PH is anomalously high was challenged by Nozaki and
Tanford (766), who measured PH 109 cm/s in phospho-
lipid vesicles and estimated the true value to be 5
1012 cm/s. Deamer and Nichols (227) argued that these
measurements were limited by development of a diffusion
potential. Diffusion potentials can be avoided by allowing
counterionflux (114). Thefinding that several cells haveundetectably small PH (185, 1054) suggests that proton
permeability is not a general property of cell membranes.
Another source of variability may be differences be-
tween conductance and permeability measurements. Ra-
dioactive tracers reveal unidirectionalflux, whereas elec-trical currents reflect only net flux, i.e., the differencebetween the unidirectional fluxes. For example, at EHthere is no net H current, but there still can be large
bidirectionalfluxes. Hence, permeability estimates basedon fluxes may be higher than electrical estimates madenear EH. On the other hand, if H
current is measured
during a large driving voltage, fluxes will be practicallyunidirectional, so the two estimates should be reasonably
consistent.
It has been suggested that both the high apparent PHand the pH independence of GH might be the result of
proton accumulation near the negatively charged phos-pholipid head groups at the membrane-solution interface
(342). In this view, PH is high because its calculation
assumes the bulk solution concentration and neglects the
possibility that the local concentration of protons at the
membrane surface may be proportionally much higher
than other cations, due to the closer approach of H3O
than a hydrated cation to the negatively charged mem-
brane. It has been known at least since 1937 that negative
surface charges tend to lower the surface pH, by up to 2
pH units in physiological solutions (215, 378, 988). Numer-
ous studies indicate that negative surface charges can
concentrate protons and other cations near membranes,
resulting in higher conductance than expected from bulkconcentrations (32, 214, 531, 716). HigherPHis measured
in negatively charged phospholipid membranes (764).
Furthermore, because the negative charges at the surface
are essentially fully screened by protons, the local proton
concentration is relatively independent of bulk pH, and
thus the apparent insensitivity of protonflux to bulk pH isalso explained (342).
A fundamental difficulty with measuring PHis that inthe physiological pH range, the [H] is up to 106 smaller
than that of other cations. Because the calculation ofPH
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effectively normalizes the measuredflux according to thenominal [H], any error is magnified, and the error isamplified at higher pH. At least in electrical measure-ments, most errors tend to increase the apparent PH. In
alveolar epithelial cells studied by voltage clamp in solu-
tions lacking small ions, PH 104 cm/s, even assuming
that the entire leak is carried by H
(242). In fact, theleakcurrent was insensitive to pH and the leak reversalpotential did not change in a direction consistent with H
selectivity, thus PH 104 cm/s by direct electrical
measurement and any proton permeability was too small
to detect (242). Similar observations were made in my-
elinated nerve (440). Also consistent with a low PH, large
changes in apical pHo do not change pHi in alveolar
epithelial monolayers (510). From the viewpoint of a cell
trying to maintain homeostasis, any proton leak is unde-
sirable. In light of the 104 increase in PH that occurs
when the cell membrane is depolarized and H channels
open, the background level of proton leak is negligible for
most purposes.It is questionable whether the traditional permeabil-
ity coefficient PH is useful for H flux through either
membranes or most channels. The Goldman-Hodgkin-
Katz (GHK) model (368, 444, 456) assumes that perme-
ation is a simple process that occurs at a rate proportional
to the rate that the permeant ion species encounters the
membrane, which in turn is proportional to the bulk
concentration.PH is thus predicted to be a constant that
is independent of pH, and lowering the pH by one unit
should increase the H flux (orgH) 10-fold. In fact, devi-ations from this prediction are more the rule than the
exception. To the extent that simple membrane H
con-ductance is independent of [H] (226, 395, 396, 755), the
parameterPH, far from being constant, increases 10-fold/
unit increase in pH. The PHof Golgi membranes increases
3.4-fold/unit increase in pH (153). PH calculated in alveo-
lar epithelial cells during maximal activation of H cur-
rents increases 5-fold/unit increase in pH (166, 242).
This type of behavior demonstrates that these systems do
not operate within the assumptions built into the GHK
permeability equations, and hence, permeability calcula-
tions have little meaning. In contrast, for gramicidin PHis
constant over a wide pH range; i.e., the single-channel H
conductance increases 10-fold/unit decrease in pH (Fig.
13). This counter-example suggests that the pH depen-dence ofPH in other systems does not reflect something
peculiar about the diffusion of protons to membranes, at
least at pH 5. Instead, it more likely indicates that a
rate-limiting step in the permeability process is slower
than the diffusional approach of protons to the mem-
brane. In the case of voltage-gated proton channels, per-
meation through the channels is thought to be rate deter-
mining (166, 234, 238 240, 242245). The GHK equationsprovide a valuable frame of reference by predicting the
behavior of a simple system. However, in the frequently
occurring situations in which PHdepends strongly on pH,
the parameterPH is not a meaningful way to evaluate or
compare protonfluxes.
2. Transient water wires
A transient water wire might occur if, due to thermalfluctuations, a chain of water molecules happened to alignacross the membrane (225, 228, 755). Although fatty acid
monolayers and cell membranes present a significant bar-rier that slows water diffusion by 104 (34, 147), water
can permeate most cell membranes, and several waters
might follow the same path once a trailblazer has led the
way. A hydrogen-bonded chain of water molecules inter-
calated between membrane phospholipids might be imag-
ined to conduct protons. A membrane-spanning chain
would need to be 20 water molecules long, and the Born
energy cost of forcing a proton into the bilayer might be
reduced by virtue of partial hydration by nearby waters
(730). The protonflux could be independent of pH if therate-determining step were the breaking of hydrogen
bonds between neutral waters, which might initiate the
turning step of the hop-turn mechanism (730) (see sect.
IIID). A recent modification of this idea is the translocationof protons by small clusters of water molecules in the
membrane (405).
There are some difficulties with the transient waterwire proposal. Although water permeability varies 27-fold
in different synthetic membranes (309), and PH varies
100-fold in different membranes, there is no correlation
betweenPH and water permeability (396). Molecular dy-
namics simulations indicate that the free energy barrier toformation of a water wire in a membrane is 108 kJ/mol,
and thus the likelihood of a membrane-spanning pore
forming is very low (658). The lifetime of such a water
wire was 10 ps in this study (long enough to transport
no more than one proton) and averaged 36 ps in a later
simulation study (1038). The H flux calculated for thismechanism could be made to agree with experimental
estimates only by assuming that a proton permeates in-
stantaneously and that the entry rate of protons into the
water wire is 108 faster than provided by diffusion (658).
Furthermore, simulations of H permeation through op-
timal water wires indicate that 100 ps is required for H
to permeate a 30-channel (120), which is longer than thepredicted lifetimes of the transient water wires (658,
1038). The mean interval between H permeation events
through gramicidin during the largest H currents re-
corded through any ion channel (2.2 109 H/s in gram-
icidin at 160 mV and 5 M HCl) (207) is 455 ps, which may
or may not represent the maximum conduction rate (see
sect. IVA4). A spontaneous water wire would have to be
narrow and transient, because otherwise other ions might
permeate (730), violating the observation that PH is 106
greater than that of other ions (755). Paula et al. (797)
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reported thatPHdecreased from 102 to 104 cm/s as
the bilayer thickness was increased from 20 to 38, andconcluded that protons were conducted via transient wa-
ter wires in thin membranes and by a solubility-diffusion
mechanism in thicker membranes. As pointed out by
Deamer (225), if PH measured in biological membranes
was found to be lower than in model (5) membranes, thenthe latter would be poor models, because biological mem-
branes may have a variety of additional transport mech-
anisms that would, if anything, increase H flux. If waterwires conduct protons across ordinary cell membranes,
then they do so at a rate that is negligibly low compared
with the proton fluxes that occur when voltage-gatedproton channels are active (242).
3. Weak acid or base shuttles
Protons can cross membranes via weak acids or
weak bases that act as proton carriers (106, 169, 671). It
has been suggested that contaminant weak acids mightaccount for the high PH reported in phospholipid bilayer
membranes (396). The weak acid mechanism has long
been recognized (486) and is illustrated in Figure 2. When
a weak acid is added to the extracellular solution, the
protonated form (HA) will be present at a concentration
determined by its pKa and the pH as described by the
Henderson-Hasselbalch equation (415, 425). The proton-
ated form can permeate the membrane far more readily
than the anionic form (A), and thus the predominant
result will be entry of HA down its gradient into the cell.
Once inside, HA will dissociate into A and H, to an
extent determined by pHi. The net result is that protons
have been transported into the cell and released there,
thus increasing pHoand decreasing pHi. The addition of a
weak base will have the opposite effect. Again, the neutral
form is far more permeant, but when B, a weak base,
enters the cell, it leaves its proton behind, lowering pHo,
and once inside the cell it will tend to bind H thus
increasing pHi. The neutral form of the acid or base willcontinue to diffuse across the membrane until its concen-
tration is the same inside and outside the cell.
A corollary to this mechanism is that weak acids and
bases tend to equilibrate across membranes according to
the pH on each side, which is important for determining
intracellular drug concentrations (e.g., Refs. 233, 443,
744). This mechanism has been exploited as a way to
estimate the pH inside cells or organelles (e.g., Refs. 152,
703, 1045). Another application of this phenomenon is the
NH4 prepulse technique (850), which is a standard
method to study pHi recovery from an acid load. This
principle has been exploited to regulate pHiin cells underwhole cell voltage clamp (242, 248, 387). One can estab-
lish a known NH4 (or triethylammonium, for example)
gradient by including a known concentration in the pi-
pette solution, and then adjusting the NH4 in the bathing
solution. Ideally, the NH4 gradient will establish an equiv-
alent H gradient. For example, 5 mM NH4 in the bath
and 50 mM NH4 in the pipette (and thus in the cell) will
lower pHi by 1 unit relative to pHo.
Because of their exquisite sensitivity to pH, voltage-
gated proton channels are effective pH meters that can be
used to report pH changes (237). Adding NH4 to the bath
produces intracellular alkalinization, which greatly dimin-ishes H currents (473). Conversely, addition of sodium
lactate or sodium acetate to the external solution rapidly
and effectively acidifies the cytoplasm, enhancing voltage-gated proton currents (473, 710).
As a practical consideration, if one wants strict con-
trol over pHi, one must worry about the presence of weak
acids or bases in the solutions. Obviously, small mole-
cules with pKanear ambient pH (e.g., HCO3, NH4
, etc.)
are perilous, but even larger molecules with pKa2 units
from ambient may produce significant changes in pHi bythe proton shuttle mechanism. For example, N-methyl-D-
glucamine (NMDG), a commonly used large imper-meantcation with pKa9.6, can cause significant shuntingof the pH gradient by the shuttle mechanism (938).
Whether it does so quickly enough to affect H currents
in a patch-clamped cell has not been reported, but devi-
ations ofVrevfromEHappear somewhat greater in a study
using NMDG solutions (232) than tetramethylammo-
nium solutions in the same cells (166). Tetrabutylammo-
nium is sufficiently lipophilic to permeate cell mem-branes (233) and has been shown to enhance proton flux(764).
FIG.2. Diagram illustrating the effects on local pH when weak acids(A) or weak bases (B) are present. The neutral form of each moleculetypically is many orders of magnitude more permeant than the chargedform. If the acid or base is present on one side of the membrane, theneutral form will permeate and change the pH on both sides of themembrane. The protonated weak acid, HA, carries its proton across themembrane and then may dissociate inside the cell, lowering intracellular
pH (pHi) and increasing extracellular pH (pHo). These pH changes willbe buffered, and the extent of the change will depend on geometricalconsiderations. The deprotonated weak base will permeate, in effectleaving a proton behind, and will tend to pick up a proton inside the cell,increasing pHi and lowering pHo.
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by a HBC mechanism (see sect. IIID), the turning step of
the hop-turn mechanism arguably might be considered a
conformational change. However, ions probably interact
with normal ion channels during permeation, and it is
possible that conformational changes in the protein (in-
duced by the presence of the ion) must occur before
conduction can proceed. Second, the rearrangement ofhydrogen bonds required during the turning step may be
subtle and hardly qualifies as a conformational change.Finally, the distinction of carriers from channels based on
the conformational change criterion is invoked to explain
the lower turnover rate of carriers. In fact, the participa-
tion of a protonatable residue at the entrance to several
proton channels has been shown to increase the effi-ciency of proton conduction (see sect. IVN). In summary,
the term channel is appropriate.
Voltage-gated proton channels exhibit gating: repro-
ducible time- and voltage-dependent activation and deac-
tivation of H current. Excess current fluctuations thatreflect stochastic opening and closing transitions, i.e.,gating, have been observed (168, 236, 720). Demonstra-
tion of the existence of gating is often presented asprov-ing an ion channel mechanism. Whether carriers mightexhibit behavior interpretable as gating is unclear. By this
criterion, voltage-gated proton channels are ion channels.
As the defining property of voltage-gated channels,including proton channels, gating is a major feature that
distinguishes channels from other types of transporters. A
channel without gating is simply a pernicious hole in a cell
membrane. In contrast, the activity of carriers (porters
and pumps) is mainly regulated by substrate availability,
and secondarily by biochemical modulation. Carriers canperform their physiological functions without a clear re-
quirement for gating. Specifically, porters and pumpshave no correlate of the full open state of ion channels, in
that at no time in their reaction cycle is there a continuous
pathway for the ion across the membrane. The open state
enables channels to have high turnover rates, whereas
carriers must undergo conformational changes during
each transport cycle.
Voltage-dependent gating must be distinguished from
voltage-sensitive flux. Any process that results in netcharge translocation across a cell membrane must in
principle be voltage sensitive. The ionflux will depend onthe driving force (596), which includes the electrical po-tential difference (voltage) across the membrane. Simple
diffusion of ions across membranes is voltage sensitive,
and so must be ionic flux through porters and pumpswhose stoichiometry of ion movement is unbalanced, so
that net charge translocation occurs. Well-known exam-
ples include the Na-K pump (833), the Na/Ca2 ex-
changer (540), a Na/HCO3 cotransporter (848), the H-
dependent glucose transporter (947), and many H/amino
acid transporters (103, 875). The ion transport rate varies
with voltage because each cycle of the carrier delivers net
charge across the membranes electric field. Even if thecharge-transferring steps are not rate limiting, the overall
process must still be voltage sensitive because voltage
will affect the probability that the transporter exists in
states immediately adjacent to the rate-limiting step (596).
However, the voltage sensitivity may not be very obvious
for a particular measurement. For example, the currentgenerated by the H-ATPase in Neurospora changed less
than twofold over 300 mV (377). The translocation of
electrons across the membrane by NADPH oxidase is
nearly voltage independent over a 150-mV range (252). In
a model of pump currents, Hansen et al. (410) showed
that the current-voltage relationship could be flat ornearly so over a wide voltage range, but steep at other
voltages. Voltage gating, in contrast, implies a discontin-
uous process: a clear difference in the mode of operation
of the transporter protein at different voltages. In the case
of voltage-gated ion channels, the probability of being
open or closed (conducting or not) depends on mem-
brane voltage. Channel gating may reflect a conforma-tional change in the protein or, in some cases, occlusion
of the conducting pathway. For all voltage-gated chan-
nels, gating is stochastic: the probability of being open or
closed depends on voltage. The current through an open
ion channel is voltage sensitive, generally increasing as
the voltage is increased relative to the reversal potential.
It can be argued that carriers and pumps must func-
tion to a variable degree of effectiveness and that this is
equivalent to the gating of ion channels; that is, carriers
may also exist in states of low functional probability,
which are incapable of reacting with the substrate. This
circumstance is obvious when a noncompetitive inhibitoris present, but can in principle occur under less well-
defined conditions, for which the termlazy-statebehaviorhas been coined (411), corresponding to theclosed ionchannel. If we could look at individual carriers, as we can
at individual channel molecules, we should see these
noncycling intervals (C. L. Slayman, personal communi-
cation). However, thus far it has been impossible to mea-
sure transport through individual carrier molecules
(whose maximal currents would be in the attoampere
range), so direct demonstration of this phenomenon is
lacking. It has been proposed for the Foproton channel of
H-ATPase (1046), that the interaction between Trp241
and His245 comprises agate.Protonation of His245 at lowpH allows interaction with Trp241, which by conforma-
tional changes or pKashifts, as speculated, allows protons
to enter the channel and access the crucial Asp61 (see
sect. IVF). The term gate has also been applied to bacte-
riorhodopsin (the best understoodactivetransporter) ina similar sense, to describe the conformational change in
the Schiff base that causes protonflux to be unidirec-tional (969). In both of these cases, however, the distinc-
tion from channels remains, because an open H channel
allows continuous H flux across the membrane down its
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electrochemical gradient, which does not occur during
the normal functioning of bacteriorhodopsin, F-ATPases,
or any other carrier-type protein.
The only conceivable alternative descriptor, carrier,
is inappropriate (see sect. IIIC). Ion carriers (uniporters)
must be voltage sensitive, because at least one form (ion-
bound or ion-unbound) must carry net charge across themembrane. As a result, a large applied voltage may trap
the carrier at one side of the membrane, and hence trans-
port will not be sustained. The resulting transient current
has been described for mutant forms of voltage-gated K
channels (R365H and R368H) in which His shuttles pro-
tons across the membrane (960, 961). The elegant analysis
of possible outcomes of histidine scanning studies of the
voltage sensor of K channels by Starace and Bezanilla
(960) distinguishes between carriers and channels. A pro-
tonatable His acting as a carrier binds a proton at one
membrane surface, moves during voltage-dependent gat-
ing to a new position in which the protonated His is
exposed to the other membrane face, and then releasesthe proton. The result is sustained H current that is
maximal near voltages where Popen is 0.5, i.e., where the
probability of gating transitions is maximal (Fig. 3C). In
contrast, mutants in which His becomes accessible simul-
taneously to both membrane surfaces act as H channels,
providing a continuous pathway for protons to cross the
membrane. This proton channel turns out to be gated
because only in one conformation, whose probability of
occurrence is voltage dependent, is the His accessible to
both sides of the membrane. In this case, the gH has a
normal sigmoid voltage dependence like other voltage-
gated channels. As shown in Figure 3, A and B, the H
current increases monotonically with voltage over a range
of 400 mV in native voltage-gated proton channels. Thisbehavior is channel-like.
One objection to the term channel is based on the
miniscule single-channel conductance. Traditionally (444,
596), channels, carriers, and pumps are characterized as
having distinctive maximum turnover rates: 105108, 102104, and 101103 s1, respectively. Although it is reason-able to argue that finding a turnover rate much higherthan the typical range suggests an erroneous classifica-tion, the same logic does not apply to a smaller-than-
typical turnover rate. If a putative carrier translocated 107
ions/s, one might suspect that it was in fact a channel.
However, if a channel conducts only 104
ions/s, this justmeans it is a channel with a low conductance. In the case
of H channels, the permeant ion normally is present at
concentrations 107 M. The H conductance of the
gramicidin channel is the largest of any ion channel at
very low pH (see sect. IVA), but extrapolated to pH 7 (see
sect. IVP) is smaller than that estimated for voltage-gated
proton channels.
FIG. 3. Absence of saturation of voltage-gated H currents contrasted with nonmonotonic voltage dependence ofcarrier-mediated H currents.A : H currents are illustrated for pulses from a holding potential (Vhold) of60 mV, in20-mV increments up to 380 mV, at pHo pHi 7.0 in a human eosinophil studied in permeabilized-patchconfiguration, as generally described in Ref. 246. The pulse duration was reduced at larger depolarizations to avoiddepletion of cytoplasmic protonated buffer, which nevertheless occurred during some pulses as evident from the droopof the current. The records with heavier lines were recorded at a faster time base (calibration bar on left). This cell hadbeen stimulated with PMA (phorbol ester) and then treated with diphenylene iodonium. B: H current-voltage relation-ship from the data in A indicates no hint of saturation (V. V. Cherny and T. E. DeCoursey, unpublished data). C:nonmonotonic current-voltage relationships at three pHi in a K
channel mutant (R365H) that acts as a proton carrier.The current disappears at large positive or negative voltages because a form of the carrier is pinned at one side of themembrane. [From Starace et al. (961). Copyright 1997, with permission from Elsevier Science.]
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D. Hydrogen-Bonded Chain Conduction
Proton permeation through a narrow channel or
through a protein is generally considered to occur by a
mechanism different from the permeation of other cat-
ions, just as proton conductance in bulk water differs
from that of other cations. Myers and Haydon (723) ex-plained the anomalously large proton conductance
through the gramicidin channel by a Grotthuss mecha-
nism of protons hopping across the row of water mole-
cules inside the channel. The gramicidin channel is
known to be a narrow pore occupied by a dozen or so
water molecules in single file (611). Protons can alsopermeate channels that do not contain a continuous row
of water molecules.
Lars Onsager explicitly proposed what has become
known as the hydrogen-bonded chain (HBC) mechanism
in 1967 (778, 779). He proposed that ions (778), including
protons (779781), might cross biological membranesthrough networks of hydrogen bonds formed betweenside chains of amino acids in membrane proteins. This
mechanism was abandoned as a means of cation perme-
ation except for the special case of protons (276, 471, 733,
734). The unique properties of protons make it possible to
devise a pathway through a membrane protein that is not
a water-filled pore like traditional ion channels (276, 733,900). Nagle and Morowitz (733) considered in detail the
properties and nature of proton conduction via a HBC.
The HBC may comprise water molecules, side groups of
amino acids capable of forming hydrogen bonds, or a
combination of the two. Amino acids suggested as poten-
tial HBC elements include Ser, Thr, Tyr, Glu, Asp, Gln,Asn, Lys, Arg, and His (734). Zundel (1102) has measured
large proton polarizability, which he considers to indicate
facilitation of proton transfer, in Tyr-Arg, Cys-Lys, Tyr-
Lys, Glu-His and Asp-His hydrogen bonds. Conduction
across a HBC occurs by migration of defects or faults.
Bjerrum (96) described two classes of defects in ice:
orientational and ionic. Two main types of orientational
faults can occur as a result of rotation of one water
molecule through 120 (Fig. 4). A Bjerrum D (doppelt double) defect occurs when two neighboring water mol-
ecules are oriented with two protons between them. A
Bjerrum L (leer vacant or empty) defect occurs when
the oxygens of two adjacent waters point toward each
other. These orientational defects can propagate throughthe ice crystal (Fig. 4C). Two types of ionic defects occur
in ice when H3O and OH are formed. These ionic
defects migrate by means of proton jumps. Various other
defects in ice have been proposed (490).
The general features of HBC conduction are illus-
trated in the diagram in Figure 5. Proton conduction
occurs in two obligate steps, called thehop-turn mech-anism. The hopping step reflects the movement of theionic defect, whereas the turning step reflects the propa-gation of a Bjerrum L fault from the right (distal) side of
the channel back to the left side. In Figure 5A, a proton
enters the HBC from the left, and through a series of
jumps, all of the protons in the chain advance, and theterminal proton exits into the solution at the distal end of
the channel. The proton that exits is not the same one that
entered, but the net result is that one proton disappears
from the proximal solution and one proton emerges into
the distal solution. A distinctive feature of HBC conduc-
tion illustrated in Figure 5B is that after the hoppingstep depicted in Figure 5A, the chain is oriented differ-
ently than before, such that another proton cannot enter
the chain from the left. First, it is necessary to reorient the
entire chain, which in the example shown is accom-
plished by rotation of each hydroxyl group. Presumably,
the hopping and turning steps of this hop-turn mecha-nism occur sequentially. A consequence of the hop-turnmechanism is that an empty proton channel has amem-ory of the last proton to permeate, which persists untilthe turning step is complete. Another consequence is that
in the absence of a membrane potential or other orienting
factor, an approaching proton has only a 50% chance that
the HBC will be oriented correctly.
FIG. 4. Formation of Bjerrum orientational faults inice.A: normal orientation of water molecules with hydro-gen bonds (implicit) between each oxygen and a hydrogenof a neighboring molecule. B : rotation of one water mol-ecule results in two types of orientational faults: the Ddefect where two hydrogens point toward each other, andthe L defect where there is no hydrogen between twooxygens.C: illustrates the infrequent occasion when theseorientational defects separate and then migrate throughthe ice crystal. [Redrawn from Bjerrum (96).]
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An intriguing aspect of the HBC conduction mecha-
nism is that the net translocation of one proton across the
membrane does not result in the translocation of one full
elementary charge. Part of the charge is translocated
during the turning step (781). In ice, the hopping step
translocates 0.64e,and the remaining 0.36eis translocated
during the turning step (782, 885); in gramicidin the cor-
responding values are 0.69e and 0.31e (901). The reorien-
tation of hydrogen bonds within the HBC results in a net
charge movement within the membrane, acting as a ca-
pacitive load. Both processes are favored by an appropri-
ate electrical driving force (i.e., positive on the side from
which proton flux originates). It would be intriguing todevise an experiment to demonstrate that H conduction
occurs in these two steps.For various reasons, it generally has been believed
that in HBC conduction the hopping step is faster than the
turning step, by an order of magnitude or more (733, 734,
810, 814, 816, 818, 819, 900). With a few notable early
exceptions (84, 366), the rate-determining step in H con-
duction in water is considered to be the reorientation of
water molecules rather than proton hopping (184, 448,
535, 818). If the turning step were also rate determining
for H current through voltage-gated proton channels,
then it would be more reasonable to consider H conduc-
tion in terms of the voltage-driven HBC reorientation
rather than proton hopping.
Although the idea that protons permeate channels via
a HBC mechanism has become widely accepted, some
have questioned whether the concept is overused. Citing
the example of ATP synthases that are driven by translo-
cation of Na instead of H, Boyer (112) suggested that
the hydronium ion, H3O, may be the transported species.
The HBC concept was proposed before any proton chan-
nel structure was known. Over the past two decades,
specific proposed proton pathways have generally pro-gressed from being mostly amino acids side groups (e